Protein modification studies

The extensive use of HNB-Br in protein modification studies has evoked much interest in its chemistry, and several reports have appeared from different laboratories 236, 253, 378). The utility of the reagent clearly depends on a thorough understanding of the mechanism of its reaction with tryptophan residues in proteins. 

Despite the complex nature of the reaction of HNB-Br with the indole nucleus of tryptophan, the reagent has been extensively used in protein modification studies to assess the role of tryptophan residues... 

Sulfenyl halides possess several particularly valuable features for protein modification studies. Their high selectively for tryptophan in proteins lacking SH-groups is especially remarkable. Furthermore, even if cysteine residues are present, selectivity for tryptophan can still be achieved since the disulphides formed by reaction of the halide with cysteine SH-groups can be easily reconverted to cysteine by subsequent reduction with mercaptoethanol. Other techniques for tryptophan modification are less specific and in several cases the reaction products are undefined. 

We are attempting to understand the biological significance of the large variations in frequency of putative LDRs, whether between different types of bacteria or archaea, or between pro- and eukaryota. We have carefully studied the literature of more than 90 example proteins selected from our disordered protein databases and found reports on the functions of most of the disordered regions (Dunker et al., 2002). The observed functions and the number of examples in each functional class are given in Table VI. As indicated, four major functional classes were found molecular recognition, molecular assembly or disassembly, protein modification, and entropic chains. 

Fowles LF, Beck E, Worrall S, et al.The formation and stability of imidazolidinone adducts from acetaldehyde and model peptides. A kinetic study with implications for protein modification in alcohol abuse. Biochem. Pharmacol. 1996 51 1259-1267. 

Ethylenimine may be used to introduce additional sites of tryptic cleavage for protein structural studies. In this case, complete sulfhydryl modification is usually desired. Proteins are treated with ethylenimine under denaturing conditions (6-8 M guanidine hydrochloride) in the presence of a disulfide reductant to reduce any disulfide bonds before modification. Ethylenimine may be added directly to the reducing solution in excess (similar to the procedure for Aminoethyl-8 described previously) to totally modify the —SH groups formed. 

Radioiodination is the process of chemically modifying a molecule to contain one or more atoms of radioactive iodine. Early studies on protein modification determined that iodine in... 

Virtually all of what is known about the secondary and tertiary structure of tubulin has been gleaned from a limited number of spectroscopic and chemical modification studies. Failure to obtain crystals of suitable quality for X-ray diffraction studies likely results from both heterogeneity in the subunits and the propensity of tubulin to polymerize into many polymorphs (see Section III). George et al. (1981) reported that as many as 17 distinct protein peaks may be discerned after isoelectric focusing of purified tubulin. 

The use of MALDI-MS analysis alone to study in vivo protein modifications has several limitations, especially when it comes to identifying the specific amino acid... 

The resolving power of mass spectroscopy is further exemplified in posttranslational modification studies of acyl carrier protein from Mycobacterium tuberculosis. Researchers were able to determine both the nature of the modification and also the mechanism by which the protein was regulated (Fung et al., 2001, Reference 7). 

We have demonstrated that this insect cell-free protein synthesis system is one of the most effective protein synthesis systems among those based on animal extracts (2). Furthermore, it has the potential to perform eukaryote-specific protein modifications such as protein W-myristoylation and prenylation (3, 4). Thus, we expect that the insect cell-free protein synthesis system will be a useful method for target protein production in the reverse chemical genetics era, as well as for postgenomic studies. In this chapter, we describe standard protocols to synthesize proteins of interest using the insect cell-free protein synthesis system. 

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